The gill epithelium of euryhaline fishes undergoes comprehensive molecular and cellular remodelling in response to salinity changes. This leads to a reversal of the direction of gill ion transport. Correlative mRNA and protein expression data strongly suggests that a switch in the dominating gill Na,K-ATPase alpha-subunit isoform, from alpha-1a in freshwater (FW) to alpha-1b in seawater (SW) is among critical factors during salinity acclimation of rainbow trout (Oncorhynchus mykiss). Accordingly it is currently hypothesized that the alpha-1b is essential for ion secretion whereas alpha-1a is the isoform driving ion uptake. However, direct functional evidence is lacking. This study sought to provide direct evidence for the functional role of alpha-1b in SW. We designed a translational vivo-morpholino to knock-down alpha-1b in the gill of rainbow trout. In an initial experiment, we injected alpha-1b vivo-morpholinos intraperitoneally in FW trout (n=4-6) followed by a 3 day SW-challenge. Vivo-morpholino injection induced a ~5% drop in muscle water content after SW-transfer, when compared with sham-injected SW-fish that were fully recovered. Knock-down reduced the SW-induced increase in gill Na,K-ATPase protein and enzyme abundance by 52 and 38 %, respectively. In the alpha-1b morpholino fish alpha-1a mRNA was increased 30-fold compared to SW-sham, suggesting compensatory stimulation of the isoform. This preliminary experiment suggests that vivo-morpholino knock-down in salmonids may be an important genetic method to determine direct functional roles of specific proteins. Further in vivo knock-down studies of gill Na,K-ATPase subunits is currently underway to substantiate and elaborate our understanding of the gill enzyme.